Our overall goal is to identify and characterize new molecular mechanisms of integrin biological functions and intracellular signaling. Integrin receptors and the extracellular matrix proteins to which they bind play crucial roles in a wide variety of cellular interactions that are important in human embryonic development, health, and disease. Integrins mediate or regulate cell adhesion, migration, and matrix assembly, as well as cell surface control of the cytoskeleton, gene expression, and growth. The spatial organization and coordination of cellular structures associated with integrins, such as adhesion sites, fibrillar extracellular matrices, the intracellular cytoskeleton, and signal transduction complexes, are thought to play central roles in embryonic craniofacial development and differentiated tissue function. We are addressing the following general questions: 1. How do integrins and the cytoskeleton interact, how are their interactions regulated, and how do they mediate cell migration and morphogenesis? 2. What molecular mechanisms initiate and modulate integrin signaling and its specificity? 3. How do cells assemble a fibronectin-based extracellular matrix? 4. What determines the type of cell-matrix adhesions that a cell forms, and are adhesions and cellular functions different in cell culture versus in three-dimensional (3D) matrices characteristic of in vivo microenvironments? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent and dimerizing chimeras, fluorescence time-lapse microscopy, and mutagenesis. Transmembrane interactions between extracellular matrix molecules, integrins, and the cytoskeleton are important for cell adhesion, shape, and migration. We have generated a variety of fluorescent molecular chimeras of cytoskeletal proteins, including vinculin, paxillin, and tensin, in an ongoing program to analyze their functions in integrin-mediated processes. In collaborative studies, the cytoskeletal proteins vinculin and talin were found to function in the transduction of force at sites of initial integrin-cytoskeletal complexes. Tensin functions instead in the unidirectional translocation of integrins during the generation of fibrillar matrices of fibronectin. Ongoing studies on mechanisms of formation of very early integrin-cytoskeletal interactions, cell adhesions, fibronectin matrix, and the roles of contractile proteins in these processes are currently in progress. A particularly well-studied mediator of integrin cytoskeletal and signaling interactions is the scaffold protein termed focal adhesion kinase (FAK). A novel regulatory role for FAK tyrosine phosphorylation in controlling its own intracellular localization was discovered. Enhanced phosphorylation of FAK unexpectedly induced its translocation out of focal adhesions, whereas FAK with lower levels of phosphorylation remained in focal adhesions. Src family kinases were required for this regulation of FAK targeting, suggesting a model in which Src kinases regulate FAK localization through phosphorylation of a specific C-terminal tyrosine residue. A direct approach to test for the roles of cytoskeletal protein interactions in adhesive and other processes is to perform gene knockout or knockdown experiments. In a collaboration with the Adelstein laboratory at NHLBI, studies of mouse embryonic stem cells and embryos with experimentally induced deficiencies in myosin II revealed an unexpected critical role in cell adhesion for only one particular isoform, myosin IIA. In the absence of myosin-IIA, cell-cell interactions were defective, which resulted in major tissue disorganization in vivo. In vitro, cells failed to remain tightly adherent to each other and were continually shed from aggregates of embryonic stem cells. The aggregates did not remain as discrete spheroids, but instead spread out unusually rapidly on substrates. These effects in vitro and in vivo were associated with defective localization of components of the major E-cadherin cell-to-cell adhesion system. These findings link myosin to cell adhesion. Further studies on the role of myosin isoforms in integrin-based migration are in progress. Integrins collaborate with growth factor receptors in initiating a wide variety of downstream signal transduction cascades. Although protein kinases have been characterized extensively in these pathways, a theoretically equally important form of regulation involves the dephosphorylation of proteins by specific phosphatases. We found that an evolutionarily conserved site in the beta-1 integrin cytoplasmic tail (tryptophan 775) functions through local regulation of the activity of a specific phosphatase, protein phosphatase 2A (PP2A). Mutation of this site results in local activation of PP2A and a resultant dephosphorylation of Akt/protein kinase B without effects on other integrin signaling. In ongoing studies, we have recently discovered that this highly specific integrin pathway strongly regulates the key cytoskeletal regulator Rac. We are characterizing the roles of Rac in regulating the intrinsic directionality of cell migration in two-dimensional versus three-dimensional tissue culture environments. A separate collaborative study examined the relationship of Rac to the formation of cell processes and migration in human keratinocytes, where glycogen synthase kinase-3 was found to regulate Rac translocation to the leading edges of cells. The importance of phosphatases in regulating these types of cell surface processes was further shown in a collaboration to study phosphorylation of another class of cytoskeletal protein. Dephosphorylation of members of the cortical cytoskeletal ERM family was found to regulate the cell surface architecture and polarity of lymphocytes. Our studies have required the development of new methods for the quantification of fibronectin matrix assembly and the signaling protein Akt, so we have provided detailed protocols in two methods papers. We also continue to provide unique research materials to many extramural researchers. For example, during this fiscal year, we completed 50 formal NIH Material Transfer Agreements to provide the research tools we have developed to researchers worldwide. In summary, our Section is elucidating mechanisms by which integrins target specific signaling pathways and organize adhesion complexes between the cell surface and the cytoskeleton involved in cell adhesion and migration. Such analyses of the mechanisms of integrin function are important for understanding how these adhesion receptors control cell movements, growth, and development. We will continue to search for novel mechanisms and modulators. Knowledge of these basic processes should facilitate creative approaches to therapy, particularly tissue engineering.